Ethanol has profound effects on the central nervous system (CNS) including pathophysiological sequelae resulting from glial cell activation. Microglia, as the resident immune cells of the brain, have been implicated in neuroinflammatory processes that occur from chronic ethanol exposure. Emerging evidence now suggests, however, that microglia can exhibit activation phenotypes other than a pro-inflammatory state depending on dose and time of ethanol exposure. Based on recent proteomic analyses of ethanol-treated microglia, we demonstrate that a significant portion of the ethanol-induced proteome response in microglia can be attributed to changes in the activity of KDM5B, a histone demethylase that catalyzes the removal of tri-methylation on Lys 4 of histone H3 (H3K4me3). Moreover, we have strong preliminary data that show ethanol induces histone methylation changes both in vitro and in vivo and that experimental modulation of KDM5B activity affects histone methylation status and subsequent pro-inflammatory response of microglia. Therefore, we hypothesize that (a) methylation of H3K4me3 and its potential impact on the overall histone methylation code is an important epigenetic mechanism that influences ethanol-induced activation of microglia and (b) changes in KDM5B-mediated histone methylation promotes the exposure time-dependent transition of microglia to a pro- inflammatory phenotype. In order to test our hypothesis, we will 1) determine ethanol-induced changes in the histone methylation code and related impact on microglial activation phenotype upon genetic and pharmacological modulation of KDM5B activity in vitro and 2) characterize the ethanol dose- and time- dependent role of KDM5B on chromatin structural and functional changes related to microglial activation phenotype in vivo. In Aim 1, unbiased, mass spectrometry-based approaches will be utilized in order to determine the impact of KDM5B activity on the histone methylation code and on the activity of epigenetic writers such as methyltransferases. Additionally, we will accurately define activation phenotype driven by histone methylation through a novel bioinformatics approach developed by our lab. In Aim 2, we will employ mass spectrometry, ChIP-Seq and computational approaches to determine chromatin functional and structural consequences related to ethanol-induced histone methylation changes in vivo. This project will be the first of its kind to accurately classify activation phenotype of microglia through novel proteomics and bioinformatics- based approaches in order to better understand microglial functional changes that occur during chronic ethanol exposure. Moreover, this project will clarify the role of the histone demethylase, KDM5B, in histone methylation changes that regulate ethanol-induced microglial phenotype, potentially allowing for the development of novel epigenetic therapies for the treatment of CNS dysfunction resulting from alcohol abuse.